The influenza virus poses a significant threat to global public health, and preemptive immunization by vaccination remains the most effective way to limit the impact of the virus. Vaccines against influenza comprise immunogenic material and may include one or more types of viral protein, including for example hemagglutinin (HA), neuraminidase protein (NA), or other viral material. One significant challenge for vaccination against influenza, however, is the frequent mutation rate of the virus.
Two key features of influenza vaccines directly result from the frequent mutation of the influenza virus. First, because multiple strains of influenza may circulate in a given season, modern vaccines are multivalent—that is, they contain antigens from multiple different influenza strains in order to confer immunity to multiple strains of the virus. Multivalent vaccines typically include HA and other viral proteins such as NA from three (trivalent) or four (quadrivalent) strains or subtypes. Current trends in vaccine development are towards developing higher valency vaccines, so as to confer the broadest possible immunity to those vaccinated. Vaccines with a valency as high as 7 have been reported. As a result, a modern vaccine may comprise a complex mixture of materials including agents for enhancing immune response (adjuvants), additives for preventing degradation of the formulation (excipients) and multiple varieties of HA and other immunogenically active proteins (e.g., NA).
The second feature of influenza vaccines that results from regular mutation of the influenza virus is that a new vaccine is generally developed each year. A strain predicted to dominate circulation in the upcoming season most often differs sufficiently from those of previous years to nullify the immunity conferred by prior vaccines, despite multivalency. In fact, a strain predicted to dominate circulation in the upcoming season (and so a component of the corresponding vaccine) may be different (e.g., a different subtype). For example, in one year a quadrivalent vaccine may be composed of influenza A HA subtypes 1 (H1) and 3 (H3) and two lineages of influenza B such as Yamagata lineage and Victoria lineage, while in the following year influenza HA subtypes 2 or 5 may be included instead of or in addition to H1 or H3. Influenza vaccines can thus vary significantly from year to year.
These characteristics of the seasonal flu vaccine impose enormous costs on the vaccine development process, in particular in quantifying the concentration of a new vaccine and its subcomponents, and in evaluating the stability of a new vaccine formulation. These costs represent open opportunities for innovation in developing improved quantification methods for the vaccine industry.
Desirable features for protein quantification in the vaccine industry include: being high-throughput and multiplexed to enable separate quantification of each of the components of a multivalent vaccine in a single test, being robust to changes in the target antigen so as to eliminate the requirement of corresponding changes in the reagents to adapt to seasonal HA mutations, and being capable of quantifying the extent of degradation of each component of a vaccine.
Currently, the only FDA- or WHO-approved method for protein quantification in influenza vaccines is the single radial immunodiffusion (SRID) assay. While widely adopted as a gold standard method, it provides none of the above mentioned desirable features: SRID is a low-throughput, singleplex method that requires new reagents each year to quantify antigen(s) in the current vaccine and is unable to adequately measure degradation. Also, SRID is a time- and labor-intensive assay that requires 2-3 days to complete and a minimum of 6 hours hands-on time by highly trained analysts. In addition, SRID is an expensive method, with costs of materials and labor for SRID analysis calculated to be approximately $1,000 per batch of 6 samples. SRID also employs subjective methods for readout and yields highly variable results. As a result, SRID is universally recognized as a significant bottleneck in the influenza vaccine development process, as evidenced by the FDA in, “A Strategic Plan: Advancing Regulatory Science at FDA” (2011). It is thus evident that SRID is not an adequate method for protein quantification in the vaccine industry.
One example method that significantly improves upon SRID is a microarray-based quantification method described in U.S. Patent Application Publication No. US2013/0494802 to InDevR, Inc. (2012). While this method is a much simpler and lower cost method than the SRID assay, it requires new reagents each year to detect and quantify antigen(s) in the current year's vaccine, and so it is not primarily concerned with variation or mutation in the target antigen. It also is not specifically designed to quantify protein degradation.
Others have attempted to provide robustness to mutations in the target antigen by using universal capture antibodies. Universal capture antibodies to HA are antibodies that bind to a highly conserved region of the HA and are thus expected to bind to most or all subtypes of HA, and most or all mutations of HA. For example, International Patent Applications PCT/IB2012/057235 to Novartis AG (2012), PCT/CA2009/000283 by Li, et al. (2009), and PCT/US2010/034604 to Sea Lane Biotechnologies (2010) describe various forms of universal antibodies and their uses. These capture agents are robust to seasonal changes in the target antigen and can be useful in the quantification of monovalent bulk material. Universal antibodies are severely limited, however, because by definition they are incapable of differentiating between strains and therefore cannot be used to quantify subcomponents of a multivalent vaccine. They therefore are of limited use for modern vaccine development and quality control. Universal antibodies also do not provide a method for quantifying degradation.
A similar, but more limited example of an attempt at universality is described in International Patent Application PCT/GB2012/052164 to Health Protection Agency (2012), which discloses methods for creating and using cross-reactive antibodies with quasi-universal binding. These antibodies are disclosed for use in singleplex quantification of potency of attenuated influenza vaccine (which consists mainly of attenuated whole virus, not free HA protein) using various singleplex methods such as ELISA, surface plasmon resonance (SPR) or the cumbersome SRlD assay. The cross-reactive antibodies described by Hufton exhibit universal binding to a sub-group of HA, for example all HA belonging to a single phylogenetic group. However, these cross-reactive antibodies are not used to quantify more than one subcomponent of a multivalent vaccine in a single assay, and thus are not practical for use in quantifying trivalent or quadrivalent vaccines. The antibodies and methods described in the application also cannot be used to detect or quantify degradation of a vaccine.
In a related prior art example, International Patent Application PCT/US2001/028877 to Medimmune, Inc. discloses an in vitro, singleplex method for measuring the immunogenicity of vaccines based on virus-like particles that is intended to overcome the challenges of determining immunogenicity by immunizing mice. In the method a vaccine, having unknown fractions of an immunogenically active epitope and an immunogenically inactive epitope, is first immobilized directly and without use of a capture agent onto a solid support. The vaccine is subsequently labeled in this immobilized and non-native state by two antibodies, each labeled by a different fluorophore, one of which binds to the immunogenically active epitope of the immobilized vaccine and the other of which binds to the immunogenically inactive epitope of the immobilized vaccine. Quantification of the relative amount of immunogenically active epitope of the vaccine in its immobilized and non-native state is used as a predictor of vaccine immunogenicity. This method is limited, however, in that it requires that a vaccine have both immunogenically active and inactive epitopes. This method is further limited because it requires that the vaccine, having unknown fractions of immunogenically active and inactive epitopes, be immobilized directly and without use of a capture agent onto a solid support prior to quantification of the fractions of immunogenically active and inactive epitopes. The immobilization of a protein directly and without use of a capture agent onto a solid support is well known to denature a significant fraction of the protein, so that subsequent quantification of the relative fraction of denatured and native protein as the method requires is rendered inaccurate. Because the relative amounts of immunogenically active and inactive epitopes in the vaccine are initially unknown in this method, the deleterious effects of immobilizing the vaccine directly and without capture agent onto the solid support cannot be accounted for in the measurement by calibration. Therefore the method described in PCT/US2001/028877 to Medimmune, Inc. is inaccurate. In addition, the pair of monoclonal antibody labels described bind to the epitopes with high specificity and could not be used to quantify immunogenicity of a different vaccine, including for example a seasonally-mutated variation of the same antigen if the seasonal mutation had altered the binding epitope significantly, as is common. The method disclosed in PCT/US2001/028877 to Medimmune, Inc. is further limited because it is a singleplex method in which the vaccine sample is immobilized on a single substrate and multiple labels are bound in the same region, prohibiting the multiplexing of the method to enable quantification of the immunogenicity of the subcomponents of a multivalent vaccine in a single reaction. Thus the method described in PCT/US2001/028877 to Medimmune, Inc. is not practical for use in the modern vaccine industry.
Thus, there is a significant need for improvements to the technology for quantification of antigen(s) in vaccines. In particular there is a significant need for a multiplexed quantification method that is able to differentiate and quantify sub-components of multivalent vaccines, that is able to quantify antigen(s) in the current annual vaccine without requiring different reagents be used for each possible mutation (e.g., seasonal), and that is able to measure degradation of the sample without the need for a reference, non-degraded sample. This new protein quantification method should also be more reliable and simpler to perform than the current SRID method, with comparable or better accuracy and precision.